Cytotoxic heteromeric protein combinatorial libraries

ABSTRACT

A method is provided for constructing, identifying and using new therapeutic or diagnostic proteins capable of binding to a target cell. The new proteins are derived by mutating a binding subunit of a wild type heteromeric cytotoxic protein to create a library of microorganism clones producing mutant proteins which are then screened for their ability to specifically bind to and kill a target cell.

CROSS REFERENCE TO RELATED APPLICATION

This application a 371 of PCT Patent Application CA98/01137, whichclaims priority from Canadian patent application number 2,222,993, filedFeb. 4, 1998, which is pending.

FIELD OF THE INVENTION

The invention relates to methods for identifying new therapeutic ordiagnostic proteins capable of binding to a target cell, and uses forthose methods.

BACKGROUND OF THE INVENTION

Most present-day chemotherapeutic agents used in controlling eukaryoticcell proliferation (as exemplified by anticancer and antifungal agents)tend to be small molecules that are able to perform a single taskrelatively well, i.e., killing or arresting the proliferation of rapidlydividing cells. Unfortunately, most of these chemotherapeutics possessminimal tissue specificity and non-optimal biodistribution profiles. Inaddition, the use of cytotoxic or cytostatic drugs in doses sufficientto halt the growth of malignant cells represents a selection pressurethat can lead to the appearance of drug resistance mechanisms.

Many plant and bacterial toxins represent successful protein designsable to penetrate mammalian cells and localize themselves intointracellular compartments. These proteins are very effective atdeleting target cells or at activating non-lethal cellular processes.The understanding of how such proteins are constructed has increaseddramatically in recent years.

A large number of plant and bacterial toxins can be grouped under acommon theme of structural organization. They are heteromeric in naturewith two or more polypeptide domains or subunits responsible fordistinct functions (1). In such proteins, the two or more subunits ordomains could be referred to as A and B, and the toxins as AB_(x) toxinswhere x represents the number of identical or homologous B subunits inthe toxin. This family of framework-related toxins includes examplessuch as Shiga and Shiga-like toxins, the E. coli heat-labileenterotoxins, cholera toxin, diphtheria toxin, pertussis toxin,Pseudomonas aeruginosa exotoxin A (2,3) as well as plant toxins such asricin and abrin. Based on their ability to block protein synthesis,proteins such as Shiga and Shiga-like toxins as well as ricin, abrin,gelonin, crotin, pokeweed antiviral protein, saporin, momordin,modeccin, sarcin, diphtheria toxin and exotoxin A have been referred toas ribosome-inactivating proteins (RIP).

SUMMARY OF THE INVENTION

The present invention utilizes the concept of using a multi-taskingheteromeric protein toxin such as Shiga toxin or other relatedribosome-inactivating protein (RIP) as a molecular template indeveloping powerful cytotoxic agents having the ability to bindspecifically to target cells. By modifying residues affecting only thereceptor-binding specificity of the toxin template, it is possible inaccordance with the invention to use the toxic A toxic domain or subunitis present in all mutant toxins as a molecular search engine inscreening combinatorial protein libraries of the toxin's template tofind mutant toxins that kill specific cells or cell types.

The inventors have thus developed a method for identifying cytotoxicmutant proteins with different receptor-binding specificity than thewild-type toxin by selecting a heteromeric protein toxin, generating alibrary of microorganism clones producing variant protein toxins byincorporating mutations into the DNA encoding for the binding subunit ofthe toxin, and screening the library against a population of screeningcells by isolating clones or pools of clones producing the variantprotein toxins, treating preparations of the population of screeningcells with the variant protein toxins produced by the clones or pools ofclones, and selecting a cytotoxic mutant protein or pool of cytotoxicmutant proteins that inhibits or kills the population of screeningcells. In preferred embodiments, the mutations may be incorporated intothe binding subunit by use of a combinatorial cassette method or bymeans of a unique site elimination method.

In one preferred embodiment, the library thus comprises geneticallyengineered bacteria or bacterial supernatants containing the variantprotein toxins. In another preferred embodiment, the library is made upof genetically engineered yeast or yeast supernatants containing saidvariant protein toxins.

The toxin may, for example, be selected from a group comprisingprokaryotic or eukaryotic proteins or protein fusion constructs capableof blocking protein synthesis. In preferred embodiments, the toxin isselected from a group comprising Shiga toxin, Shiga-like toxins, ricin,abrin, gelonin, crotin, pokeweed antiviral protein, saporin, momordin,modeccin, sarcin, diphtheria toxin and Pseudomonas aeruginosa exotoxinA. In further preferred embodiments, the binding subunit is derived fromthe B-subunit template of either Shiga toxin or related Shiga-liketoxins, or homologous counterparts from E. coli heat labileenterotoxins, cholera toxin, pertussis toxin or the receptor bindingdomain of ricin. The target cell may be a tumour cell, for example, abreast cancer cell.

Thus in one embodiment of the invention, it has been shown that a familyof related mutant combinatorial toxins, from, for example Shiga toxin orShiga-like toxin 1, can be derived that can kill breast cancer cellswhich were previously insensitive to the native toxin.

The invention also provides a method of killing or inhibiting a targetcell by treating the target cell with a cytotoxic mutant protein or poolof proteins selected by the methods of the invention. The target cellmay be the same as the cells used as the population of screening cells,or it may be a different type of cell which shares a common receptorwith the screening cells to which the cytotoxic mutant protein binds.Thus, as noted below, cytotoxic mutant proteins identified using ascreening cell population may be further screened against cells from apatient using methods known in the art.

In another embodiment, the invention provides a method for identifyingtherapeutic proteins having binding specificity for a target cell byselecting a heteromeric protein toxin, generating a library ofmicroorganism clones producing variant protein toxins, screening thelibrary against the population of screening cells by the methods of theinvention, and then testing for effectiveness against the target cells(if the target cells are different form the screening cells). Tominimize non-specific toxicity, the cytotoxic mutant proteins arefurther screening against non-target cells to select a therapeuticmutant protein or pool of therapeutic mutant proteins that are lesseffective at inhibiting or killing the non-target cells than atinhibiting or killing the target cells.

The invention further teaches a method for constructing diagnosticprobes for detecting the presence of a cell surface marker by selectinga mutant heteromeric protein toxin by the screening methods of theinvention, selecting from the library of microorganism clones a clonewhich is producing the cytotoxic mutant protein, preparing a diagnosticDNA sequence by incorporating a marker DNA encoding for a detectablemarker into a binding subunit DNA sequence in the selected clone and,generating diagnostic probes from the diagnostic DNA sequence. In apreferred embodiment, the marker DNA codes for green-fluorescent protein(GFP).

The invention also teaches methods for constructing a medicament havingbinding specificity, for example, by selecting by the methods of theinvention the cytotoxic mutant protein, selecting from the library ofmicroorganism clones a clone which is producing the cytotoxic mutantprotein, preparing a medicament DNA sequence by incorporating medicinalpolypeptide DNA encoding for a medicinal polypeptide into a bindingsubunit DNA sequence in the selected clone and, generating a medicamentfrom the medicament DNA. The medicaments of the invention may be usedfor treating a condition requiring targeting a medicine to a target celloccurring in a host organism.

In other embodiments, the invention provides kits useful for performingthe methods of the invention, the kits including a selected heteromericprotein toxin and suitable supports useful in performing a method of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the amino acid sequences of the A subunit (FIG. 1A;corresponding to SEQUENCE ID. NO. 1) and B (FIG. 1B; corresponding toSEQUENCE ID. NO. 2) subunit of Shiga-like toxin 1.

FIG. 2 shows backbone representations of Shiga toxin (ShT; panel A, sideview) and its B subunit (panels B and C, bottom view).

FIG. 3 is the oligonucleotide sequences of Primer A (FIG. 3A;corresponding to SEQUENCE ID. NO. 3) and Primer B (FIG. 3B;corresponding to SEQUENCE ID. NO. 4) synthesized for creation of the ShTlibraries.

FIG. 4 is a graph showing cytotoxicity curves showing the ability of ShTvariant 506 to kill SK-BR-3 cells on passage 34 (♦), 40 (▪), 56 (▴), and68 (▾); and for the effect of native ShT on passages 40 (□), 56 (Δ), and59 (◯).

FIG. 5 is a graph showing the difference in cell viability observed whenSK-BR-3 cells were exposed to a 14 nM solution of either the native ShT(●) or the ShT variant 506 (◯) at various cell passage numbers.

FIG. 6 is a graph showing the ability of three ShT variants (nativetoxin (Δ); ShT variant 122 (♦); ShT variant 126 (●); ShT variant 824 (▪)identified which kill CAMA-1 cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present inventors have constructed protein combinatorial librariesbased on the structural template of a heteromeric protein toxin for usein deriving toxin mutants with receptor specificities directed attargets resistant to the native toxin. The strength of this new approachstems from the fact that all members of these libraries are cytotoxic innature. This common property of all toxin variants can thus be used as asearch engine in finding mutants in these libraries with new receptorspecificity. The screening strategy involves the use of simple cellcytotoxicity assays, which immediately identify optimized therapeutic aswell as diagnostic agents, thus eliminating the need to redesign anylead compounds to enhance their cellular uptake, intracellularprocessing and/or cytotoxicity.

Heteromeric plant and bacterial toxins have a structural organizationwith two or more polypeptide domains or subunits responsible fordistinct functions, referred to as A and B. The toxins may be referredto as AB_(x) toxins where x represents the number of identical orhomologous B subunits in the toxin. This family of framework-relatedtoxins includes examples such as Shiga and Shiga-like toxins, the E.coli heat-labile enterotoxins, cholera toxin, diphtheria toxin,pertussis toxin, Pseudomonas aeruginosa exotoxin A (2,3) as well asplant toxins such as ricin and abrin.

Based on their ability to block protein synthesis, proteins such asShiga and Shiga-like toxins as well as ricin, abrin, gelonin, crotin,pokeweed antiviral protein, saporin, momordin, modeccin, sarcin,diphtheria toxin and exotoxin A have been referred to asribosome-inactivating proteins (RIP). The potency of RIPs is exceedinglyhigh; one molecule of diphtheria toxin A chain (99) or ricin A chain(100) having been shown to be sufficient to kill a eukaryotic cell. Thecrystal structures for many of these molecules have now been established(4-12), and insights into their functions have mostly focused on theidentification of residues involved in the catalytic activity of Achains and on mapping B subunit residues involved in receptor-bindingactivity.

The data presented herein support the broad potential of combinatorialShiga toxin libraries (or libraries of any heteromeric, cytotoxicmember) as sources of potentially cell-specific cytotoxic and diagnosticagents. Since the receptor binding potential of combinatorial proteinssuch as Shiga toxin (B-subunit pentamer) can be dissociated from itscytotoxic A subunit, the present invention also provides a method fordeveloping non-cytotoxic, diagnostic probes for detecting the presenceof useful cell surface markers to aid in the selection of therapeuticstrategies. Furthermore, because the binding unit of combinatorialproteins is genetically discrete from the toxin subunit, selected mutantbinding units can be produced independently of the toxin subunit, andcan be incorporated with marker DNA or therapeutic DNA to creatediagnostic probes or cell-specific therapeutic proteins.

The invention thus provides a method for identifying and producingtherapeutic or diagnostic proteins capable of binding specifically to atarget cell, which said proteins are derived from a wild typeheteromeric protein having a cell surface binding domain or subunit anda cytotoxic domain subunit, comprising the steps of: a) creatinglibraries of mutant heteromeric proteins in which the cell bindingdomain or subunit has been randomly mutated; and b) screening thelibrary using the cytotoxic domain present in all mutant toxins as abuilt-in search engine against a population of screening cells which islacking or has lower levels of receptors which cause sensitivity to thewild type protein, and identifying those mutants which kill thescreening cells. As used herein, the term “substantially insensitive”refers to cells useful as screening cells as a result of such a lack ofreceptors recognized by the wild-type toxin, or the presence of asufficiently reduced level of such receptors that the activity of thewild-type toxin and the mutant toxin can be distinguished.

The invention also provides a method for constructing and screeningtherapeutically useful toxin variants that will bind to surface markers(glycolipids, glycoproteins, or proteins, as examples) expressed onhuman tumour cells in preference to normal cells. The invention furtherteaches a method for constructing and screening toxin variants whichtarget a defined eukaryotic cell populations such as pathogenic fungi orwhich can be used to control the growth of rapidly proliferating cells(implicated in scar management, tissue remodelling, or skin diseases forexample). Further, the invention teaches a method for constructing andscreening therapeutically useful non-cytotoxic, diagnostic probes fordetecting the presence of useful cell surface markers to aid in theselection of therapeutic strategies. Shiga toxin variants cansubsequently be modified by dissociation of the variant from itscytotoxic subunit or by inactivation of the variant's cytotoxic subunit,or the DNA encoding for the binding subunit of selected variants can beused to construct various diagnostic or therapeutic tools.

The construction of heteromeric protein toxin libraries allows thoseskilled in the art to rapidly identify new cytotoxic/diagnostic probeswith altered receptor targeting properties. The procedure is exemplifiedherein with reference to Shiga toxin and Shiga-like toxin. Since thenatural receptor for the B subunit of Shiga toxin is a glycolipid, thespecificity of mutant B subunits derived from Shiga libraries harbouringa low level of degeneracy in the sequence of its loops (which arestructures implicated in receptor specificity) may be directed at uniquecarbohydrate structures located on glycoproteins or glycolipids. In thecase of toxin libraries containing highly degenerate sequences withinthe two loop regions known to mediate binding, it is expected that thepotential surface structures recognized will be very diverse. As in thecase of antibody combining sites, B subunit variants may bind to aspectrum of molecular entities such as proteins, peptides, nucleic acidsor even organic moieties rather than to sugars or glycolipids.

The construction of cytotoxic heteromeric libraries offers severaldistinct advantages. Firstly, the libraries are permanent and can beindefinitely screened to provide a continual source of new therapeuticor diagnostic agents. Secondly, the lethal character of the resultingtoxin mutants towards eukaryotic cells allows one to easily screen foruseful constructs having a specificity for unique cell targets (such ascancer cells). Thirdly, useful mutant B subunits can be generated in theabsence of a cytotoxic A chain, permitting the immediate creation ofnon-cytotoxic diagnostic agents that can be used to detect the presenceof unique markers on cell types in either in vitro or in vivo settings.

A person skilled in the art will appreciate that the methods of thepresent invention can be applied to immunotoxins and related growthfactor-toxin conjugates to develop multi-tasking agents able to providemore guided therapies or to be utilized as diagnostic tools for cancerand other patients.

For example, concerning therapeutic tools, the present inventionpresents a method for identifying therapeutic proteins having bindingspecificity for a target cell for the purpose of developing novelpeptide or protein drug delivery vehicles and targeting systems. Havingselected an appropriate heteromeric protein toxin having a toxic subunitand a binding subunit, the methods taught herein and adapted, ifnecessary, by means known in the art, may be used to generate a libraryof microorganism clones producing variant protein toxins, byincorporating mutations into the binding subunit DNA encoding for thetoxin in a microorganism. The library is then screened by methods suchas those set out above to select clones or pools of clones producing thecytotoxic mutant proteins which inhibit or kill a population ofscreening cells. The selected cytotoxic mutant proteins may optionallybe further screened against cells from a patient using methods known inthe art, by treating preparations of such cells with clones or pools ofclones producing cytotoxic mutants, and selecting a cytotoxic mutantprotein or pool of cytotoxic mutant proteins that are effective atinhibiting or killing target cells and are safe for the patient.

As another example, the toxic domain or subunit could be modified orreplaced with another toxic domain or subunit, selected or engineeredsuch that the toxin requires a co-factor or the like to be activated. Inthis manner, the therapeutic protein may be administered to a host, andafter sufficient time has passed to allow the therapeutic protein toadhere to the target cells, the cofactor can be introduced to the host,thus killing or inhibiting the target cells.

Concerning the construction of diagnostic tools, the methods of theinvention provide for selecting a heteromeric protein toxin andgenerating a library of microorganism clones producing variant proteintoxins from the heteromeric protein toxin, for the screening andselection of a mutant toxin with enhanced sensitivity and selectivity.The library is then screened against a population of screening cells bythe methods of the invention, i.e. by isolating clones or pools ofclones producing the variant protein toxins, treating preparations ofthe population of screening cells with the variant protein toxins andselecting a cytotoxic mutant protein or pool of cytotoxic mutantproteins that inhibits or kills the population of screening cells. Ifdesired to alleviate toxicity, for example where the selected diagnostictool is to be used in vivo, one skilled in the art may modify thecytotoxic mutant protein or pool of proteins by dissociation of thebinding subunit from the toxic subunit or by inactivation of a toxicsubunit of the cytotoxic mutant protein. One may additionally, ifneeded, label the cytotoxic mutant protein or pool of proteins with adetectable marker. Alternatively, the genes producing the cytotoxicmutant protein or pool of proteins are manipulated to endogenouslyproduce detectable markers. For example, the invention is used toconstruct diagnostic probes for detecting the presence of a cell surfacemarker by first identifying by the methods taught herein a cytotoxicmutant protein or pool of proteins, and subsequently preparing adiagnostic DNA sequence by incorporating, by any means known in the art,a marker DNA encoding for a detectable marker into the binding subunitDNA sequence(s) of the cytotoxic mutant protein or pool of proteins, andgenerating diagnostic probes from the diagnostic DNA sequence. Examplesof detectable markers known in the art include various enzymes,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable proteins include horseradish peroxidase, variantsof green fluorescent proteins, luciferase, alkaline phosphatase oracetylcholinesterase. Examples of suitable fluorescent materials includeumbelliferone, fluorescein, dansyl chloride or phycoerythrin. An exampleof a suitable luminescent material includes luminol. Examples ofsuitable radioactive materials include P-32, S-35, Cu-64, Ga-67, Zr-89,Ru-97, Tc-99m, In-111, I-123, I-125, I-131, Re-186 and Au-199. Theproteins may also be labelled or conjugated to one partner of a ligandbinding pair. Representative examples include avidin-biotin andriboflavin-riboflavin binding proteins. Methods for conjugating orlabelling the proteins discussed above with the representative labelsset forth above may be readily accomplished using conventionaltechniques.

The invention further presents a method for treating a conditionrequiring targeting a medicine to a target cell occurring in a hostorganism. This may be accomplished by use of the methods set out abovein selecting a therapeutic protein having binding specificity andsubsequently modifying the therapeutic protein by conjugating a medicine(such as a toxin) to the binding subunit of the protein to form apeptide/protein drug delivery vehicle, and administering same to a hostorganism having a disease associated with the target cell an effectiveamount of that drug. In the use of this invention to treat a condition,one skilled in the art may optionally further modify the therapeuticprotein by the various methods discussed herein.

Further, the invention comprises kits to assist one in carrying out themethods of the invention. Reagents suitable for applying the methods ofthe invention may be packaged into convenient kits providing thenecessary materials, and packaged into suitable containers, optionallycontaining suitable supports useful in performing the methods of theinvention.

Mode of Action of Shiga and Shiga-Like Toxins

Shiga toxin (ShT) and Shiga-like toxins (SLT) are structurally relatedbacterial toxins involved in the pathogenesis of bacillary dysentery,hemorrhagic colitis, the hemolytic uremic syndrome, and thromboticthrombocytopenic purpura (19-21). Shiga toxin, the first member of thisfamily of cytotoxins to be reported in 1903 (22,23) is produced byShigella dysenteriae I. Shiga-like toxins have been recently identifiedas virulence factors elaborated by enterohemorrhagic strains of E. coli(24-28). In particular, the E. coli strain O157:H7, which producesShiga-like toxin 1, has been recently identified as the causative agentresponsible for recent mass outbreaks of food poisoning in Japan and theUnited States.

Shiga (ShT) and Shiga-like (SLT) toxins possess the smallest known Bsubunit (less than 70 residues) of all AB_(x) toxins, and their Asubunit has an identical catalytic activity as the corresponding subunitin ricin. FIG. 1 shows the amino acid sequences of the A and B subunitsof Shiga-like toxin 1. Panel A (corresponding to SEQUENCE ID. NO. 1)shows the catalytic A subunit. Panel B (corresponding to SEQUENCE ID.NO. 2) shows the B subunit with the three boxed regions representingloops harbouring residues postulated to be involved in creating areceptor binding cleft for CD77.

FIG. 2 shows backbone representations of Shiga toxin (ShT; panel A, sideview) and its B subunit (panels B and C, bottom view). As seen in FIG.2, ShT and SLT-1 have identical B subunits. The catalytic A subunit (12,Panel A) has its C-terminus inserted into the central hole of the Bsubunit pentamer (14). The B subunit pentamer (14, Panel B) isstabilized by intra- and inter-subunit interfaces involving β-sheets.Two of the three loop regions of the B subunit boxed in FIG. 1 (residues15-19 and 30-33) are darkened (16) to show the orientation and locationof these loops in relation to the β-strand structure of the B subunitand the A chain itself. Loop 58-66 is located in the same vicinity asloops 15-19 and 30-33 and was not highlighted for reasons of clarity. InPanel C, each identical B subunit is shaded differently to illustratetheir symmetrical arrangement giving rise to a pentamer.

These toxins are proteins composed of six subunits; one catalytic Asubunit (293 amino acids; MW 32,317) involved in the blockage of proteinsynthesis and five B subunits (69 amino acids; MW 7600 each) necessaryfor the attachment of the toxin to cells (29-35; FIG. 2). The B subunitsspontaneously assemble into a pentamer in solution (FIG. 2, panels B andC). The structure of these toxins typifies a common motif employed byother larger bacterial toxins such as cholera toxin and the E. coliheat-labile enterotoxins (6,7) and pertussis toxin (8).

The cell specificity of ShT and SLT-1 is encoded by its B subunit whichrecognizes the glycolipid globotriaosyl ceramide (referred to as CD77 orGb₃; Galα1-4Galβ1-4Glcβ1-1 Ceramide; ref. 36,37). CD77 has a relativelylimited tissue distribution, and is expressed on a number of humancancers (13, 102-105). The native toxin has recently been shown to beeffective in purging, a human lymphoma from bone marrow (13). Followingits attachment to susceptible cells, Shiga toxin is endocytosed fromcoated pits (38-40). The A-chain is processed to a smaller 27 kDa A,fragment through a selective nicking and reduction of the native chain.The A₁ fragment is responsible for the inactivation of eukaryoticribosomes (29) acting as a highly specific N-glycosidase which cleaves asingle adenine residue from 28S rRNA (41,42). Depurination at that siteinhibits peptide elongation by preventing the EF-1 dependent binding ofaminoacyl tRNA to the 60S ribosomal subunit (43-45).

EXAMPLE 1 Designing Shiga Toxin Libraries to Derive Useful Diagnosticand Therapeutic Agents Targeted at Defined Eukaryotic Cell Populations

In accordance with the invention, the receptor specificity of the toxin,which is encoded by its B subunit, was altered by random mutagenesis.Mutations in the B subunit were kept to a minimum in order to lessen anynegative effects on other functions of the toxin such as the toxicity ofits A chain and the proper folding and assembly of the holotoxin (i.e.,pentamerization of the B subunit, insertion of the A₂ domain into the Bpentamer, exposure and orientation of the protease sensitive loop, andpacking environment of the translocation domain).

Shiga and Shiga-like toxin 1 have identical B subunits. The B subunit isa small protein composed of only 69 amino acids that pentamerizesspontaneously in solution. Its crystal structure (as a pentamer of Bsubunits) has been solved in the presence and absence of the A subunit(4,5) and has been shown to be identical in either context. Each Bsubunit monomer within the pentameric structure is composed of 6β-strands (β1, residues 3-8; β2, residues 9-14; β3, residues 20-24; β4,residues 27-31; β5, residues 49-53; β6, residues 65-68) involving 31 ofits 69 amino acids (45%; FIG. 2). A single α-helix (residues 36 to 46)accounts for 16% of the remaining structure. These elements of secondarystructure appear essential for the maintenance of the pentamer integrityand its association with the A₂ domain of the A chain (FIG. 2). Thus,any perturbations in these regions may result in folding problems. Threeloop regions composed of more than two amino acids are left. They aredelimited by residues 15 to 19, 32 to 35, and 54 to 64, respectively.Mutagenesis studies of the B subunit have indicated that substitutionsat positions 16, 17, 30, 33, and 60 either abolished or reduced thecytotoxic potential of the resulting toxin while an Asp to Asnsubstitution at position 18 altered the receptor specificity of thetoxin (85-89). Molecular modelling studies involving the docking of CD77(Gb₃) to the B subunit have implicated residues located in these loops(90,91). It has been hypothesized that there are two potential bindingsites for CD77 on the B subunit pentamer, namely, sites I and II(90,91). Residues located in regions 15-19 and 30-33, in particularAsn15, Asp 16, Asp 17, and Phe 30, form most of the putative bindingsite I (91). The calculated interaction energy derived from modellingstudies suggested that site I is likely to be the predominant sitemediating CD77 interaction (91). Thus, results from both site-directedmutagenesis and docking experiments suggest that residues found in loopregions are sites where random mutagenesis may lead to an alteredreceptor specificity. As described herein, residues are perturbed withintwo loop regions, namely, residues 15-19 (loop 1), and residues 30-33(loop 2; technically speaking, this region is not a loop but ratherrepresents the end of the β4 strand and the beginning of the secondloop). Random mutagenesis in loop 3 (residues 58-64; FIG. 2) may also beeffective in achieving the objective of the invention. Though initialstudies have focussed on the aforementioned regions of the molecule,this delimitation does not preclude the possibility of targetting any ofthe B subunit residues in attempts to alter specificity of the toxin.

Nine residues are involved in loops 1 and 2, creating a potentiallibrary complexity of the order of 20⁹ (5×10¹¹ different mutantproteins, if all nine residues were totally randomized and all potentialcombinations recovered). It is, therefore, advantageous to reduce thelevel of complexity of the toxin library so that the nine residues ofinterest are not completely randomized. This goal was accomplished bysynthesizing oligonucleotides for use in the mutagenesis procedure thathave increasing levels of nucleotide “doping”. The selection of anoligonucleotide with the desired level of doping for mutagenesissubsequently allows direct control over the level of diversity in thelibrary made from that particular oligonucleotide pool. For example,mutations at 5 amino acid positions out of 9 in the target region, wouldyield a diversity of the order of 20⁵ (3.2×10⁶ mutant toxins), a moresatisfactory level of diversity. Indeed, the screening of libraries withgreater than 10⁶ compounds has not previously proven necessary forchemical or peptide libraries in terms of identifying useful “lead”compounds (using either binding assays or functional assays in thescreening process). Additionally, the number of potential target siteson cell surfaces will be large and will increase the need for screeningsteps.

EXAMPLE 2 Mutagenesis and Construction of Heteromeric Cytotoxic ProteinCombinatorial Libraries

Shiga and Shiga-like toxin 1 differ in sequence by only one amino acidin their A subunit and have identical B subunits. Although the randommutagenesis procedures described herein use the SLT-1 gene, the simplerterminology “Shiga toxin library” has been used rather than “Shiga-liketoxin 1 library” in defining an ensemble of mutant proteins derived fromthe Shiga toxin structural template.

Briefly, the recombinant plasmid pJLB28 (32) was used as a template formutagenesis. This construct carries a BglII-BalI fragment ofbacteriophage H-19B inserted in pUC19, which specifies for theproduction of active SLT-1 holotoxin. An additional construct was madeby cloning a PCR product consisting of the SLT-1 gene carried by pJLB28into the prokaryotic expression vector pTUG (92). The latter construct,pTGXH, encodes for the production of SLT-1 with a hexa-histidinesequence fused to the N-terminus of the A chain, to facilitate thepurification of toxin variants.

There are numerous methods available for generating random mutations inDNA. Mutagenesis using synthetic oligonucleotides with regions ofdefined degeneracy (93-96) is an established and reliable techniquewhich satisfies the requirements of the invention, i.e., a rigidlydefined mutagenic window and the need to control the frequency and typeof mutations generated. Mutagenic oligonucleotides (98-mers) with thesequence indicated in FIG. 3 were synthesized on an Applied Biosystems392 DNA synthesizer. Loop 1 and loop 2 represent residues 15-19 and30-33 of the B subunit, respectively. Primer A (FIG. 3A; correspondingto SEQUENCE ID. NO. 3) was synthesized to have controlled levels ofrandomization in the two loops as described in the text. Primer B (FIG.3B; corresponding to SEQUENCE ID. NO. 4) overlaps primer A by 15 basesat its 3′ end, and was used to create a combinatorial cassette bymutually primed synthesis in conjunction with primer A. Restrictionsites used to clone the libraries are indicated in bold. The primerswere designed to mutagenize both loops 1 and 2 simultaneously. A silentmutation introducing a new Sac I restriction site between the two zoneswas incorporated into the mutagenic primer to facilitate screening oftransformant DNA and to allow for the “shuffling” of zones betweenvariants. Five different (98-mers) mutagenic primers were synthesizedwith increasing levels of “randomness” in loops 1 and 2, so thatlibraries of predictable size could be generated. This strategy wasaccomplished by synthesizing codons in the loop regions in the form“NNS”, where N is a base added to the growing chain from a mixture ofthe wild-type base “doped” with a fixed percentage of the three otherbases, and S is a base added from a 1:1 mixture of cytosine and guanine.The latter aspect of the method allows codons specifying all 20 aminoacids, but makes the chances of observing a given amino acid closer to1:20 by reducing the degeneracy of the DNA code. Also, only the amberstop codon TAG can be generated using this strategy; thus, minimizingthe production of truncated proteins.

The five mutagenic primers synthesized had doping levels ranging from1.2% to 75%, where 75% represents completely random codons (i.e., thephosphoramidite mixture used to place the given base contained 25%wild-type bases and 25% each of the other bases). A mutagenic primermade with a 12.5% doping level was chosen for initial studies to producea library where the number of potentially different sequences (3.2×10⁶mutants, or a mutation rate of approximately 5 substitutions out of 9per clone) was well within the limits of Escherichia coli transformationefficiency.

Two strategies have been employed so far to incorporate the mutagenicoligonucleotides into the toxin gene to create libraries of variantproteins; using the unique site elimination method (97) or by creating acombinatorial cassette. Single-stranded random mutagenic primers wereincorporated into double-stranded plasmids using the unique siteelimination (USE) mutagenesis method (97) employing the Pharmacia USEkit. This method allows mutagenesis to be performed on anydouble-stranded plasmid in the absence of restriction sites (97).

In an attempt to increase the efficiency of the mutagenesis procedureand to maximize the diversity of clones obtained, a combinatorialcassette method has also been employed to generate toxin libraries. Inthis method, the same oligonucleotide pools depicted in FIG. 3A wereannealed to an overlapping oligo sequence shown in FIG. 3B. Adouble-stranded cassette was created by mutually primed synthesis, i.e.,by including DNA polymerase and dNTP's in a reaction with theoverlapping pair such that each oligonucleotide would code for theformation of the opposite sense strand. The cassette was then amplifiedusing PCR and cloned directly into the vector containing the toxin geneat sites AccI and PstI.

Further refinements to the mutagenic process are known to those in theart. For example, libraries may be created using an entirelyligation-free system employing the uracil DNA glycosylase method (101).Notably, the demonstrated ability to use the same random oligonucleotidepools in a variety of different mutagenesis procedures underscores theflexibility of the system and its high capacity for adaptation and rapidimprovement.

EXAMPLE 3 Screening of Heteromeric Cytotoxic Protein CombinatorialLibrary Against Breast Cancer Cell SK-BR-3

An initial library was constructed using the USE method with a mutagenicoligonucleotide with a 12.5% doping level. Following transformation ofE. coli strain JM101 with vector DNA within which the randomizedoligonucleotide had been incorporated, colonies picked from agar plateswere grown in 96-well plates with conical well-bottoms and individualclones were picked from isolates. To confirm that the variants wereproducing toxin with an A chain capable of inactivating ribosomes,extracts produced by 17 clones selected at random were collected andassayed for their ability to inhibit eukaryotic protein synthesis. Thisassay uses Promega TnT coupled transcription/translation reticulocytelysate system, and consists of measuring the product of a luciferasegene in the presence and absence of bacterial extracts. The extracts ofall the clones tested inhibited translation of the luciferin protein.Five of these variants were sequenced, and the nucleotide sequences ofthe randomized loop regions are listed in Table 1. The tested clonesreflected the desired rate of mutation of approximately 5 out of 9 aminoacid changes per clone.

Table 1—Nucleotide and Amino Acid Sequences of ShT Mutant Clones andWild-Type Shiga Toxin

Clone Loop 1 Loop 2 Mutation Rate Wild-type AAT GAT GAC GAT ACC Seq IDNo 5 TTT ACC AAC AGA Seq ID No 6 N D D D T Seq ID No 7 F T N R Seq ID No8 ShT 6 AAC GAG GAG GAG ACG Seq ID No. 9 TTC GCG AAC AGC Seq ID No 115/9 N E E E T Seq ID No 10 F A N N Seq ID No 12 ShT 13 AAC GAG CAG GACACC Seq ID No 13 TTC ACC CAC AGG Seq ID No 15 3/9 N E Q D T Seq ID No 14F T H R Seq ID No 16 ShT 15 AAG GAG AAC GAG AGC Seq ID No 17 TTC GCG AACAAC Seq ID No 19 7/9 K E N E S Seq ID No 18 F A N N Seq ID No 12 ShT 17AAG GAC GAC GCG AGG Seq ID No 20 TTG ACC CAG AGG Seq ID No 22 5/9 K D DA R Seq ID No 21 L T Q R Seq ID No 23 ShT 19 AAG GAC GAC GAC ACG Seq IDNo 24 TTG ACC CAG AGG Seq ID No 22 3/9 K D D D T Seq ID No 25 L T Q RSeq ID No 23Table 1. Comparison of nucleotide and amino acid sequences betweenmutagenic loops of five ShT mutant clones recovered from one of our ShTcombinatorial libraries (12.5% doping level) and wild-type Shiga toxin.Loops 1 and 2 represent residues 15-19 and 30-33 of the B subunit of ShT(or SLT-1) respectively.

The ability of a ShT variant to kill cells represents the most directand practical measure of its utility. This function (cytotoxic propertyretained by all toxin variants) provides each mutant with a built-insearch engine allowing one to screen any ShT combinatorial librariesagainst any eukaryotic cells to identify novel mutant toxins that cankill such cells.

In one example, the breast cancer cell line SK-BR-3 is used as theinitial eukaryotic population of screening cells. SK-BR-3 cells wereobtained from the American Type Culture Collection. Cells were grown andmaintained in α-MEM media supplemented with 10% fetal calf serum. Cellswere grown at 37° C., 5% CO₂ and the media changed every 2 days. Celldensities were chosen to ensure that each cell line was at approximatelythe same degree of confluency at the beginning of a cytotoxicity assay.

Toxin-containing extracts were produced by freeze-thawing [B. H.Johnson, M. H. Hecht, Bio/Technology 12, 1357 (1994)] pellets fromovernight cultures of individual clones of E. coli strain JM101transformed with mutagenized vector DNA. The clones were grown in either200 μl (clones screened on SKBR-3) or 800 μl (clones screened on CAMA-1)of Terrific broth supplemented with 100 mg/ml carbenicillin (TB-carb).Extracts were allowed to intoxicate the breast cancer cells for 48hours, then cell viability was measured using either the tetrazoliumsalt WST-1(4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate; Boehringer Mannheim), or by measuring total cellularprotein content using the dye sulforhodamine B (SRB) [P. Skehan, et al.,J. Nat. Cancer Inst. 82, 1107 (1990)]. Sib selection [M. McCormick,Meth. Enzmol. 151, 445 (1987)] was employed when screening ShT clones.When clones that killed the target cells were identified, they wereinoculated into 3 ml of TB-carb, grown overnight at 37° C. with shakingat 250 rpm and then extracted and re-tested for cytotoxicity against thecell line.

A set of 1000 clones were picked from the (12.5% doping) library to testthe screening strategy. An 8×8 sib selection grid system (98) was used,whereby a given clone was pooled with seven others in a system whereevery clone tested was present in two separate pools. The 8-clone poolswere amplified and then extracts from the mixtures were tested forcytotoxicity on Vero cells (a cell line highly susceptible to thewild-type toxin) and the human breast cancer SK-BR-3 cell line (a cellline that is insensitive to the wild-type toxin). A colorimetric assaybased on the cleavage of the tetrazolium salt WST-1 by mitochondrialdehydrogenases in viable cells was used to quantify cell viability. Thecleavage of WST-1 gives rise to a water-soluble formazan that can bereadily measured in the visible range (450 nm) using a 96-well plateformat and a plate reader, thus allowing the use of high throughputscreening approaches. Other colorimetric cell viability assays were orcould be used such as alternate tetrazolium salts XTT, MTT, or dyes suchas sulforhodamine B. In addition, screening could be performed usingcell proliferation assays measured in terms of counting cell colonies orthe incorporation of radiolabeled nucleotides or amino acids intonucleic acids or proteins. Clones that were implicated in producingcell-killing toxins were retested individually on the same cell lines.This preliminary set of clones has yielded thus far at least 14 clonesthat show a dramatic increase in their ability to kill SK-BR-3 cellsrelative to the wild-type ShT. Several lysates were able to delete z 90%of SK-BR-3 cells in relation to control wells containing viable cells(no toxin present). Plasmid DNA was recovered and sequenced fromisolates that consistently killed SK-BR-3 cells in cytotoxicity assays.Sequence alignments in the mutated B-subunit loop regions of 14 mutanttoxins are presented in Table 2.

Several clones showed reduced cytotoxicity on Vero cells but enhancedSK-BR-3 toxicity. The latter clones are of significant interest, sincethe goal of the invention is to alter the natural specificity of thetoxin from the CD77 glycolipid to another cell surface marker. Scalingup the screen to greater than 1000 single clones optimizes the screeningstrategy.

The clones identified from this low-level degeneracy library show amarked conservation of the first loop (residues 15 to 19), which mayreflect a “skewing” of the isolates recovered towards ShT mutants ableto bind to receptor homologs of CD77. In contrast, clones picked atrandom from the same library did not show any predisposition towardmaintenance of the wild-type sequence, and had amino acid substitutionsin their target regions at the predicted rate (results not shown).Several of the cytotoxic ShT variants were overexpressed, purified tohomogeneity and assessed for their cytotoxicity against SK-BR-3 cells.

Table 2—SK-BR-3 Library

Clone Loop 1 Loop 2 wild-type N D D D T Seq ID No 7 F T N R Seq ID No. 8ShT 66 N E E E T Seq ID No 10 E F T G Seq ID No. 26 ShT 110 N D D D TSeq ID No 7 F T K S Seq ID No. 27 ShT 128 T T D D P Seq ID No 28 G T R GSeq ID No. 29 ShT 220 N D D D T Seq ID No 7 L T N G Seq ID No. 30 ShT241 N D D D T Seq ID No 7 F T K S Seq ID No. 27 ShT 256 N D D D T Seq IDNo 7 L P N R Seq ID No. 31 ShT 265 N D D D T Seq ID No 29 F T N C Seq IDNo. 38 ShT 415 K E D E S Seq ID No 33 L T K R Seq ID No. 34 ShT 506 N DD D T Seq ID No 7 L T K S Seq ID No. 35 ShT 619 Y D D N P Seq ID No 36 LT N S Seq ID No. 37 ShT 766 N D D D T Seq ID No 7 L T K R Seq ID No. 34ShT 767 K K E E P Seq ID No 38 C A N R Seq ID No. 39 ShT A22 N D D D TSeq ID No 7 L T K R Seq ID No. 34 ShT A25 N D D D T Seq ID No 7 L T N RSeq ID No. 40Table 2. Amino acid sequences of clones exhibiting cytotoxic activity onSKBR-3 cells (recovered from a 12.5% doping level library) and CAMA-1(clones recovered from a 75% doping level library). Loops 1 and 2represent the same B subunit residues indicated in Table 1.

EXAMPLE 4 Screening of Heteromeric Cytotoxic Protein CombinatorialLibrary Against Breast Cancer Cell Line CAMA-1

A second library, this time using an oligonucleotide pool with a moredegenerate doping level of 60%, was created using the combinatorialcassette method described previously. The library was screenedessentially as the first using the sulforhodamine B cell viability assayand the cell line CAMA-I as the population of screening cells. This cellline is also a breast carcinoma like SKBR-3, but has been shown to lackthe CD77 marker and is extremely resistant to the native SLT-1 toxin.CAMA-1 cells were obtained from the American Type Culture Collection.Cells were grown and maintained in α-MEM media supplemented with 10%fetal calf serum. Cells were grown at 37° C., 5% CO₂ and the mediachanged every 2 days. The cell densities were chosen to ensure that eachcell line was at approximately the same degree of confluency at thebeginning of a cytotoxicity assay.

A collection of 600 SK-BR-3 single clones from the cassette library werescreened for cytotoxic effect on CAMA-I, and as in the case of SKBR-3several promising toxin variants were identified, whose sequences areshown in Table 3. The clones identified from this highly diverse librarywere found to have amino acid sequences in the target regions which werealmost completely different from those in the wild type toxin. Thesequence diversity of this library is very large (up to 20⁹ mutants) andexceeds the transformation efficiency limits of Escherichia coli)(˜10¹⁰). Cytotoxicity curves for three ShT mutants derived from thisscreening are presented in FIG. 6. CD₅₀ values ranging from 100 to 300nM were calculated for these ShT variants (variants 122, 126 and 824;cell passage #13; symbols: native toxin (Δ); ShT variant 122 (♦); ShTvariant 126 (●); ShT variant 824 (▪). Each point represents the percentcell viability calculated from the average of experiments performed intriplicate.

Table 3—CAMA-1 Library

Clone Loop 1 Loop 2 wild-type N D D D T Seq ID No 7 F T N R Seq ID No 8ShT 122 C L L N G Seq ID No 41 Y Q E P Seq ID No 42 ShT 126 Q G L Q LSeq ID No 43 T L T G Seq ID No 44 ShT 142 T G A T M Seq ID No 45 P T G ISeq ID No 46 ShT 241 F R P A G Seq ID No 47 L R C G Seq ID No 48 ShT 308P Y V F L Seq ID No 49 M V A N Seq ID No 50 ShT 324 K S M D Q Seq ID No51 L S K W Seq ID No 52 ShT 715 Q G E Y G Seq ID No 53 I Q E R Seq ID No54 ShT 823 M V Q E K Seq ID No 55 S K K Q Seq ID No 56 ShT 824 D Y F Q TSeq ID No 57 R H Y S Seq ID No 58Table 3. Amino acid sequences of clones exhibiting cytotoxic activityCAMA-1 (clones recovered from a 60% doping level library). Loops 1 and 2represent the same B subunit residues indicated in Table 1.

EXAMPLE 5 Assessment of Cycling of Cell Surface Molecules Targeted byToxin Variants

It was found that the susceptibility of SK-BR-3 cells to the variousmutants changed as a function of cell passages. FIG. 4 depicts thisphenomenon for the toxin variant derived from clone 506. FIG. 4 showsthe effect of variant 506 on passage 34 (♦), 40 (▪), 56 (▴), and 68 (▾);and for the effect of native ShT on passages 40 (□), 56 (Δ), and 59 (◯).The passage number represents the number of passages of the SK-BR-3 cellline in culture starting at passage #24 as defined by the American TypeCulture Collection. Each point represents the percent cell viabilitycalculated from the average of experiments performed in triplicate. TheCD₅₀ for this ShT mutant ranged in values from 3.5 nM to >290 nMdepending on the passage number of the SK-BR-3 cell line. The screenthus recorded the rapid and transient nature of selected surface markerson SK-BR-3 cells even in the case of a relatively clonal population ofcells. Interestingly, the change in sensitivity of SK-BR-3 cells towardsShT-506 is a random event in relation to passage number (FIG. 5). Thetargeted cell line (starting at passage #24; ATTC) cycled from beingresistant to ShT variant 506 at passage #32, to sensitive at passages#34 and #40, to being almost resistant again by passage #56 and finallyreverting to being sensitive to the action of ShT variant 506 bypassages #63 and #68. In contrast, SK-BR-3 cells remain resistant to theaction of the native toxin over a similar range of cell passages,indicating that cell surface molecule CD77 remains stable over time. Forexample, FIG. 5 illustrates the difference in cell viability observedwhen SK-BR-3 cells were exposed to a 14 nM solution of either the nativeShT (●) or the ShT variant 506 (◯) at various cell passage numbers (eachpoint represents the percent cell viability calculated from the averageof experiments performed in triplicate).

This phenomenon suggests that the surface expression of CD77 is moreregulated than the marker recognized by ShT variant 506. Differences inthe cytotoxicity of ShT-506 toward SK-BR-3 cells were observed for morethan 100 passages of the cell line, all performed under identical growthconditions (results not shown). Screening of the libraries of thesevariants thus provided a valuable source of probes to study theexpression and rapid cycling of cell surface molecules. Other types ofstudies could exploit this feature of the libraries of the presentinvention. For example, collections of ShT variants could serve tophenotypically define differentiation events leading to the acquisitionof metastatic potential of tumor cells, or to study the development ofhematopoietic cell lineages.

The fact that most tumours are heterogeneous suggests that a largenumber of candidate toxins should be identified, perhaps to beadministered as a cocktail in therapy. This fact underscores the powerof the described approach, since a single toxin template can be screenedfor many potential specificities, whereas other agents such asimmunotoxins have specificity only for cells exhibiting their targetreceptor. The concept of specificity also assumes that the expression ofa targeted cell surface marker remains constant within a cellpopulation. Results presented in FIG. 4 would argue against the validityof this hypothesis. The results herein demonstrate the ease with whichone can identify a collection of toxin mutants cytotoxic toward arelatively homogeneous cell population by the use of this invention.Searches based on cytotoxicity assays are amenable to high throughputscreening strategies and thus may allow a more thorough exploration ofvariant toxin libraries to find such families of toxin mutants. In thecontext of ex vivo purging situations the utility of toxin variants canbe readily assessed by exposing bone marrow cells or peripheral stemcells to these agents and observing the level of reconstitution ofhaematopoietic cell lineages using flow cytometry under in vitro or invivo settings (transplantation experiments in SCID, NOD/SCID mice, forexample; ref. 14). The initial selection of breast cancer cell linesSK-BR-3 and CAMA-I as the population of screening cells used with theShT library searches stems from the fact that most autologous bonemarrow transplants (ABMTs) or peripheral stem cell transplantations arepresently performed on breast cancer patients, and that an ex vivopurging of their stem cells may prove beneficial in terms of thepatient's long-term survival (13,106-107). The requirement for auniquely selective agent for cancer cells, a major concern in the designof in vivo treatment strategies, is greatly reduced, since one or moremutant toxins may be clinically useful as long as the targeted surfacemarker is absent on human stem cells.

Analogies can be drawn between the structure of antibodies and ShTvariants in terms of their ligand binding properties. Antibodies harbourtwo antigen combining sites while ShT B subunit pentamers possess atleast five identical ligand binding domains. Both structural entitiespossess a conserved scaffold of β-strands linked by loop regions whichtogether define their receptor binding domains. As in the case ofantibody combining sites, B subunit variants may thus bind to a spectrumof molecular entities such as proteins, peptides, nucleic acids or evenorganic moieties rather than to sugars or glycolipids (such as CD77).However, the diversity of toxins derived from the libraries of theinvention is not biased by genetic recombination and somatic mutationswhich dictate antibody repertoire. The vast potential forreceptor-binding diversity present in the library highlights the factthat as the degeneracy of the library increases, so does the diversityof molecules on the cell surface available as ligands to mutatedB-subunits.

EXAMPLE 6 Use of a Mutant Toxin to Develop a Diagnostic Tool

After selecting a heteromeric protein toxin and generating a library ofmicroorganism clones producing variant protein toxins from theheteromeric protein toxin, the library is then screened against a targetcell by the methods of the invention, i.e. by isolating clones or poolsof clones producing the variant protein toxins, treating preparations ofthe target cell with the variant protein toxins and selecting acytotoxic mutant protein or pool of cytotoxic mutant proteins thatinhibits or kills the target cell. The genes producing the cytotoxicmutant protein or pool of proteins are manipulated to endogenouslyproduce detectable markers. A diagnostic probe is thus constructed fordetecting the presence of a cell surface marker by incorporating, by anymeans known in the art, a marker DNA encoding for a detectable markerinto the binding subunit DNA sequence(s) of the cytotoxic mutant proteinor pool of proteins, and generating diagnostic probes from thediagnostic DNA sequence. The present inventors have usedgreen-fluorescent protein (GFP) from the jellyfish Aequorea victoria asa fluorescent marker for such diagnostic probes. This marker is usefulin a variety of organisms ranging from bacteria to higher plants andanimals (Tsein, R Y, 1998, Annu Rev Biochem, 67:509-44; Chalfie, M., Tu,Y., Euskirchen, G., Ward, W. W., and Prasher, D. C., 1994, Science.263:802-805). Formation of the fluorescent chromophore is speciesindependent and the gene product is easily detectable by its intensefluoresence (Prasher, D C, 1995, Trends Genet, 1995, August,11(8):320-3). It is useful for monitoring gene expression in vivo, insitu, and in real time (Rizzuto R. et al, 1998, Trends Cell Biol, July,8(7):288-92). When expressed in either eukaryotic or prokaryotic cells,GFP gives forth a bright green fluorescence. GFP fluoresces in theabsence of any other intrinsic or extrinsic proteins, substrates, orcofactors. Fluorescence is stable, species-independent, and in somecases can be monitored noninvasively in living cells and whole animals(Chalfie, M., et al., supra).

In light of the demonstrated utility of the invention, a person skilledin the art will appreciate that the method can be applied to other cellswith the expectation that useful therapeutic and diagnostic moleculeswill be identified. With numerous target sites on cells, it is expectedthat a large number of mutant toxins will be found with cytotoxicactivity.

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1. A method for making a cytotoxic mutant protein or pool of proteinsfrom a cytotoxic wild type protein, said mutant protein or pool ofproteins having receptor-binding specificity for a receptor that isdifferent from the receptor to which the wild type protein has receptorbinding specificity, comprising: (A) selecting a heteromeric proteintoxin having a toxic domain or subunit and a binding domain or subunit,wherein the heteromeric protein toxin is a ribosome inactivatingprotein; (B) incorporating mutations into DNA encoding the bindingdomain or subunit of the heteromeric protein toxin to produce aplurality of variant forms of the heteromeric protein toxin; (C)generating a library of microorganism clones producing variant forms ofthe heteromeric protein toxin; (D) screening the variant forms of theheteromeric protein toxin of said library against a population ofscreening cells by (i) isolating clones or pools of clones producingsaid variant forms of the heteromeric protein toxin, (ii) treatingpreparations of said population of screening cells with variant forms ofthe heteromeric protein toxin produced by the isolated clones or poolsof clones, (iii) observing the treated preparations of said populationof screening cells for toxicity, and (iv) selecting based on theobservation of toxicity a cytotoxic mutant protein or pool of cytotoxicmutant proteins that inhibits or kills said population of screeningcells to a greater extent than the wild-type cytotoxic protein, wherebysaid selected mutant protein or pool of proteins has the differentreceptor binding specificity that is reflected by the observation oftoxicity, wherein the screening cells are insensitive to the selectedcytotoxic heteromeric protein toxin at a concentration used in thescreening; and (E) making additional copies of the selected cytotoxicmutant protein or pool of proteins.
 2. The method of claim 1, whereinthe cells in the population of screening cells are eukaryotic.
 3. Themethod as claimed in claim 1, wherein said library comprises bacteria orbacterial supernatants containing said variant protein toxins.
 4. Themethod as claimed in claim 1, wherein said library comprises yeast oryeast supernatants containing said variant protein toxins.
 5. The methodas claimed in claim 1, wherein said binding domain or subunit DNA is ina plasmid in said microorganism.
 6. The method of claim 1, wherein saidmutation is incorporated into said binding domain or subunit by use of acombinatorial cassette method comprising: (A) preparing synthetic mutantoligonucleotides capable of annealing with a corresponding wild typeoligonucleotide from said binding domain or subunit; (B) annealing saidsynthetic oligonucleotide from said binding domain or subunit to anoverlapping wild type oligonucleotide to form a double strandedsequence; (C) creating a combinatorial cassette by mutually primedsynthesis of said double stranded sequence; and (D) incorporating saidcassette into a vector containing a gene for said toxin.
 7. The methodas claimed in claim 1 wherein said mutation is incorporated into saidbinding domain or subunit by means of a unique site elimination method.8. The method as claimed in claim 1 wherein said heteromeric proteintoxin is selected from the group consisting of Shiga toxin, Shiga-liketoxins, ricin, abrin, gelonin, crotin, pokeweed antiviral protein,saporin, momordin, modeccin, sarcin, diphtheria toxin and Pseudomonasaeruginosa exotoxin A.
 9. The method as claimed in claim 8 wherein saidheteromeric protein toxin is Shiga toxin or Shiga-like toxin
 1. 10. Themethod as claimed in claim 9 wherein said mutation is incorporated intoloop regions at residues 15-19, 30-33 or 58-64.
 11. The method asclaimed in claim 9 wherein said mutation is incorporated into loopregions at residues 15-19 or 30-33.
 12. The method as claimed in claim 2wherein the cells in the population of screening cells are tumour cells.13. The method of claim 12 wherein the tumour cells are breast cancercells.
 14. The method as claimed in claim 13 wherein said breast cancercell is CAMA-I.
 15. The method as claimed in claim 1 wherein saidbinding domain or subunit is derived from the B-subunit template ofeither Shiga toxin or Shiga-like toxins, or homologous counterparts fromE. coli heat labile enterotoxins, cholera toxin, pertussis toxin or thereceptor binding domain of ricin.
 16. A method for identifyingtherapeutic proteins having binding specificity for a target cell,comprising: (A) making a cytotoxic mutant protein or pool of proteins bythe method as claimed in claim 1; and (B) screening said cytotoxicmutant protein or pool of proteins against said target cells and againstnon-target cells by treating a preparation of target and a preparationof non-target cells with said cytotoxic mutant protein or pool ofproteins, and selecting a therapeutic protein or pool of therapeuticproteins that are effective to inhibit or kill said target cells andthat are less effective at inhibiting or killing said non-target cellsthan at inhibiting or killing said target cells.
 17. A method forconstructing a diagnostic probe for detecting the presence of a cellsurface marker comprising: (A) selecting a cytotoxic mutant protein thatspecifically binds to the cell surface marker by the method as claimedin claim 1, said cell surface marker being the receptor on the targetcell population in the method of claim 1; and (B) preparing a diagnosticprobe by labeling the selected cytotoxic mutant protein in a mannerwhich maintains the ability of the binding domain or subunit of theselected cytotoxic mutant protein to specifically bind to the cellsurface marker.
 18. The method of claim 17, wherein the diagnostic probeis prepared by a method comprising: (i) preparing a diagnostic DNAsequence comprising a marker DNA encoding a detectable marker and abinding domain or subunit DNA sequence encoding the binding domain orsubunit of the selected cytotoxic mutant protein; and (ii) expressingthe diagnostic DNA sequence to generate a diagnostic probe.
 19. A methodfor constructing diagnostic probes as claimed in claim 18 wherein saidmarker DNA codes for green-fluorescent protein (GFP).
 20. The method ofclaim 17, further comprising the step of modifying the cytotoxic mutantprotein or pool of proteins by dissociation or inactivation of the toxicdomain or subunit of the cytotoxic mutant protein.
 21. A method formaking a targeted medicament for delivery to a target cell having a cellsurface marker, said targeted medicament comprising a binding portionand a medicament portion comprising the step of: (A) identifying abinding subunit which binds to the cell surface marker by a processcomprising the steps of (i) selecting a heteromeric protein toxin havinga toxic domain or subunit and a binding domain or subunit, wherein theheteromeric protein toxin is a ribosome inactivating protein; (ii)incorporating mutations into DNA encoding the binding domain or subunitof the heteromeric protein toxin to produce a plurality of variant formsof the heteromeric protein toxin; (iii) generating a library ofmicroorganism clones producing variant forms of the heteromeric proteintoxin; (iv) screening the variant forms of the heteromeric protein toxinof said library against a population of screening cells by (a) isolatingclones or pools of clones producing said variant forms of theheteromeric protein toxin, (b) treating preparations of said populationof screening cells with variant forms of the heteromeric protein toxinproduced by the isolated clones or pools of clones, (c) observing thetreated preparations of said population of screening cells for toxicity,and (d) selecting based on the observation of toxicity a cytotoxicmutant protein or pool of cytotoxic mutant proteins that inhibits orkills said population of screening cells to a greater extent than thewild-type cytotoxic protein, whereby said selected mutant protein orpool of proteins has receptor-binding specificity for the target cellpopulation that is reflected by the observation of toxicity, wherein thescreening cells are insensitive to the selected wild-type heteromericprotein toxin at a concentration used in the screening; and (v)determining the sequence of the binding domain or subunit of theselected cytotoxic mutant protein for use as the binding portion of thetargeted medicament; and (B) combining the binding portion with themedicament portion.
 22. The method of claim 21, wherein the bindingportion and the medicament portion are combined by preparing amedicament DNA sequence comprising a medicinal DNA encoding a medicinalpolypeptide for use as the medicament portion, and a binding domain orsubunit DNA sequence encoding the binding portion, further comprisingthe step of expressing the medicament DNA sequence.
 23. A method formaking a nucleic acid sequence, or pool of nucleic acid sequences,encoding a cytotoxic mutant protein, or pool of cytotoxic mutantproteins, of a cytotoxic wild type protein said mutant protein or poolof proteins having receptor-binding specificity for a receptor that isdifferent from the receptor to which the wild type protein has receptorbinding specificity, comprising: (A) selecting a heteromeric proteintoxin having a toxic domain or subunit and a binding domain or subunit,wherein the heteromeric protein toxin is a ribosome inactivatingprotein; (B) incorporating mutations into DNA encoding the bindingdomain or subunit of the heteromeric protein toxin to produce aplurality of variant forms of the heteromeric protein toxin; (C)generating a library of microorganism clones producing variant forms ofthe heteromeric protein toxin; (D) screening, the variant forms of theheteromeric protein toxin of said library against a population ofscreening cells by (i) isolating clones or pools of clones producingsaid variant forms of the heteromeric protein toxin, (ii) treatingpreparations of said population of screening cells with variant forms ofthe heteromeric protein toxin produced by the isolated clones or poolsof clones, (iii) observing the treated preparations of said populationof screening cells for toxicity, and (iv) selecting based on theobservation of toxicity a cytotoxic mutant protein or pool of cytotoxicmutant proteins that inhibits or kills said population of screeningcells to a greater extent than the wild-type cytotoxic protein, wherebysaid selected mutant protein or pool of proteins has the differentreceptor binding specificity that is reflected by the observation oftoxicity, wherein the screening cells are insensitive to the selectedwild-type cytotoxic heteromeric protein toxin at a concentration used inthe screening; and (E) making additional copies of the nucleic acidsequence or pool of nucleic acid sequence encoding the selectedcytotoxic mutant protein or pool of cytotoxic mutant proteins.
 24. Themethod of claim 23, wherein the cells in the population of screeningcells are eukaryotic.
 25. The method of claim 24, wherein the cells inthe population of screening cells are tumor cells.
 26. The method ofclaim 25, wherein the tumor cells are breast cancer cells.
 27. Themethod of claim 23, wherein the binding domain or subunit is derivedfrom the B-subunit of either Shiga toxin and Shiga-like toxins, orhomologous counterparts from E. coli heat labile enterotoxins, choleratoxin, pertussis toxin or the receptor binding, domain of ricin.
 28. Themethod of claim 1, wherein in step B the mutations are randomlyincorporated into the DNA encoding the binding domain or subunit of theheteromeric protein toxin.